Certain polyamines, such as putrescine, spermidine and spermine, have been established as biochemical markers or indicators of normal and pathological growth. In malignancy, the urinary concentrations of spermidine reflect the tumor cell loss and the urinary level of putrescine is related both to the number of tumor cells in cell cycle and to the tumor cell loss factor. A greater than two-fold increase in urinary spermidine within 72 hours of chemotherapy predicts a complete or a partial response with a high degree of accuracy. Urinary putrescine titre may be valuable not only in assessing the early response to therapy but also in determining whether the chemotherapy promotes a later burst of cell proliferation. Erythrocyte spermidine concentrations also appear to track alterations in tumor kinetics. Alterations in intracellular and extracellular polyamines in other pathologies such as psoriasis, muscular dystrophy and cystic fibrosis also accurately reflect the disease activity and, in those cases studied, response to therapy.
Therefore, the determination of polyamine concentrations in extracellular fluids and in erythrocytes allows for (1) the early assessment of response to multimodality therapy, (2) disease or tumor staging, and (3) assessment of disease activity including long-term monitoring of polyamine concentrations to pinpoint remission and relapse in adjuvant patients. Information obtained by the monitoring of polyamines could result in prolongation of survival time of patients as well as assist in the design of the most effective therapy regimen for the pathology. Since other such specific kinetic markers are not available, polyamines can be clinically utilized to track tumor evolution and tumor response to therapy in those patients at high risk in which such measurements could be translated into therapeutic efficacy.
The polyamines, putrescine, spermidine and spermine, are ubiquitous components of both procaryotic and eucaryotic cells. They function as the organic cations of the cells and serve roles in protein and nucleic acid synthesis, structure and function. Polyamines also are found in the extracellular fluids in mammalian organisms. Their levels in plasma, urine and cerebrospinal fluid are useful indicators of disease activity, specifically with relation to neoplasia, and are of predictive value in assessing the efficacy of treatment and the maintenance of remission.
Elevated levels of putrescine, spermidine and/or spermine have proven to be indicators of pathology in cancer, cystic fibrosis, psoriasis and Duchenne muscular dystrophy (DMD). Further, an increased excretion of spermidine (greater than two-fold of control level prior to therapy) prescribes a partial or complete response to therapy with a high degree (greater than 95%) of accuracy.
Current sensitive methods for estimating polyamines, accurate to the pmole level, utilize either high-pressure liquid chromatography, which requires an initial large monetary investment in equipment and is limited in the number of samples which can be analyzed in one day, or a purified specific antibody. The ability of the enzyme transglutaminase to incorporate the polyamines into a covalent isopeptide linkage with glutamine residues on protein substrates provides a novel means for estimating polyamine levels in tissue or biological fluid samples. This application presents the details of a radioenzymatic method, sensitive to the pmole level, for rapidly and simply measuring polyamines.